CA1265050A

CA1265050A - Purification of blood clotting factors and other blood proteins on non-carbohydrate sulfated matrices

Info

Publication number: CA1265050A
Application number: CA000513596A
Authority: CA
Inventors: Robert E. Jordan
Original assignee: Miles Laboratories Inc
Current assignee: Bayer Corp
Priority date: 1985-07-12
Filing date: 1986-07-11
Publication date: 1990-01-30
Also published as: ES2002098A6; JPH0724723B2; US4721572A; DE3686295T2; EP0208215B1; EP0208215A3; ATE79052T1; DE3686295D1; EP0208215A2; JPS6226227A

Abstract

Inventor: ROBERT E. JORDAN

Invention: PURIFICATION OF BLOOD CLOTTING
FACTORS AND OTHER BLOOD PROTEINS
ON NON-CARBOHYDRATE SULFATED
MATRICES

ABSTRACT OF THE DISCLOSURE

There is disclosed a non-carbohydrated sulfated matrix and the use thereof to isolate and purify blood clotting factors and other blood proteins by a procedure involving the adsorption of at least one of such factors and proteins onto the non-carbohydrate sulfated matrix followed by elution of such factors and proteins onto the non-carbo-hydrate sulfated matrix followed by elution of such factors and proteins from the matrix.

Description

5~

This invention relates to a non-carbohydrate synthetic gel matrix that is useful in a process to isolate and purify blood clotting factors and other blood proteins using affini-ty chromatographic techiyues.
Many useful blood fractions and pro-teins may be obtained from blood and blood plasma by known techniques.
Andersson et al, U.S. Patents 3,842,061 and 3,920,625, disclose a cross-lin~ed sulfated polysaccharlde gel matrix adsorbing agent and the use thereof to isolate and purify antithrombin and blood coagulation factors, respectively, from animal tissue materials such as blood, blood products or plasma fractions.
Menache-Aronson et al, U.S. Patent 4,447,416, the pro-duction of an intermediate purified non-thrombogenic factor IX concentrate containing factor IX in major therapeutic amounts and containing ~actors II, VII and X in minor, non-thrombogenic amounts.
The sulfated matrices used to date all have been based on the use of carbohydrate as the backbone. Although such carbohydrate matrices has been useful especially in laboratory-scale separations, it has become evident that, for large-scale production work, gels having greater strength and flow capacity and resistance to microbial attack would be desirable.
According to this invention, there is provided a sul-fated, synthetic, non-carbohydrate gel matrix adsorbing agent comprising a sulfatable polymer selected from a synthetic organic affinity chromatography gel matrix prepared by polymerization or copolymerization of at least one polymerizable organic monomer and from silica particles followed by sulfation of the polymer backbone by a sulfating agent. In another aspect of this invention, there is provided a process for isolating and purifying blood coagulating, or clotting, factors ~' ' .
.

and other blood proteins comprising contacting blood, plasma, plasma protein concentrates, or tissue culture fluids containing bloo~ coagulating factors or other blood proteins with -the sulfated synthe-tic affinity chromatography gel matrix a~sorbing agen-t according to this invention.
The sulfated synthetic affinity chromatography gel ma-t-rix adsorbing agent may be any organic polymeric mate-rial that is sulfatable and that is useful in affinity chromatographic separation operations. Examples of suitable synthetic gel matrices include polyhydroxylated acrylates and methoacrylates, silica particles, and polyvinylbenzene wherein the hydroxyl groups or benzene moiety can be sulfated by reaction of the hydroxyl groups or benzene moiety with a suitable sulfating agent such as, for example, sulfonyl chloride, chlorosulfonic acid and the like.
Preferably, the sulfated synthetic gel matrix according to this invention comprises a sulfated synthetic poly-hydroxylated acrylate of methacrylate or a polyhydroxysilica particle.
An especially useful sulfated polyhydroxylated acrylate gel matrix is that derived from copolymerization of the monomer, N-acryloyl-2-amino-2-hydroxymethyl-1,3-propane diol, which is commercially available under the trademark TRISACRYL~(Reactifs IBF, France).
An especially useful sulfated polyhydroxy silica particle material is that derived from sulfation of the polyhydroxy silica material commercially available under the trademark ., ~ .
.

- ---- - . : , :
- : : : ,, . :

,~ , NuGel~ P-NP (Separation Industries, Metachem, New Jersey, U.S.A.).

Any conventional procedure for sulfating an organic 5 hydroxyl group or aromatic (i.e. benzene) nucleus may be used to sulfate the synthetic gel matrix. A convenient sulfation procedure useful to sulfate the TRISACRYL and NuGel matric?s mentioned above is a modification of the method reported by Miletich et al., Analytical Bio-o chemistry, 105, 304 - 310 (1980), involving the use of chlorosulfonic acid in pyridine.

Among the blood clotting factors and other blood proteins which may be isolated and purified according to the process 15 of this invention by using the sulfated, synthetic, non-carbohydrate gel matrix of the invention are factor IX, factor X, factor II, factcr VII, F. VIII, protein C, protein S, prothrombin, tissue plasminogen activator (TPA) and the like.
The blood coagulating, or clotting, factors and other proteins thus obtained, whether from plasma or tissue culture fluids using biotechnology, can be formulaked into pharmaceutical preparations for therapeutic use. The 2s pharmaceutical preparations may be treated by known means to render them free of infectious microorganisms such as bacteria and viruses and the like, including hepatitis B
virus and the AIDS-causing agents (AXV, H~LV-III, and LAV
agents). Such treatments include sterile filtration, heat treatment in the wet or dry state, chemical treatment, ultraviolet irradiation, and treatment with colloidal silica.

The following examples illustrate but a few embodiments of 3~ the present invention and are not to be construed as limiting in scope. All parts and percentages are by weight ,., ~,.. ..
.

.

and all temperatures are in degrees Celsius unless other-wise indicated.

EX~MPLES

A. Preparation of sulfated matrices for adsorp~ion of coagulation proteins.

EXAMPLE 1.
o Pr~paration of Sulfated Trisacryl Trisacryl is a synthetic, non carbohydrate gel matrix prepared by the copolymerization of the monomer N-acryloyl-

2-amino-2-hydroxymethyl-1,3-propane diol. Trisacryl GF
15 2000 is manufactured by Reactifs IBF (France) and was ohtained as an aqueous suspension.

1200 ml of suspended Trisacryl GF 2000 was dehydrated on a sintered glass funnel by sequential washes of distilled water (5 x 1200 ml), methanol (5 x 1200 ml) and acetone (5 x 1200 ml). The acetone-containing cake was spread on an aluminum foil surface and heated with an incandescent lamp in a fume hood until the powder was thoroughly dried (14 hours). The yield of dry Trisacryl powder from this 2s treatment was 321 ~rams.

The sulfation reaction was carried out by a modification of the method previously described for dextran beads by Miletich et al (Analytical Biochemistry, 105, 304 - 310, 1980). The trisacryl powder was added with stirring to a solution of chlorosulfonic acid in pyridine ob~ained by ~he dropwise addition of 428 mls of acid to 2.14 liters of pyridine in a dry ice/ethanol bath. The solution was heated to 70 degrees C to completely dissolve the pyridinium before addition of the trisacryl powder. Once added the suspension was stirred until the powder had absorbed all of the liquid. The reaction mixture was ~ , ,:

:: , ~ ::

maintained at 70 C for 2 hours and then at 50 C for 16 hours.

The wet trisacryl c~ake was transferred to scintered glass 5 funnels on vacuum flasks attached to a water aspirator.
Remaining pyridine solution was collected and combined with the first wash effluents for disposal. The gel was washed with a total of 50 liters of 2M NaCl adjusted to pH lO.
The gel was then further washed with 50 liters of distilled water at pH lO. The final weight of the washed gel was 1580 grams.

The sulfated trisacryl gel was e~uilibrated before use in 10 volumes of a buffer consisting of 0.05 M sodium citrate pH 6.5 and 0.10 M sodium chloride. The equilibrated gel was then packed in the desired chromatographic column.

EXAMPLE 2.
Sulfation of Polyhydroxv Silica Sulfation of polyhydroxy silica (NuGel P-NP obtained from Separation Industries, Metuchen, NJ) was carried out in a similar fashion to that described for Trisacryl in Example 1 above. A quantity of dry glass beads (10 grams) was added to a solution which had been prepared by the addition of 12.5 ml chlorosulfonic acid to 65 ml of pyridine as described in Example 1. The suspension was heated at 70 C
for 16 hours after which the pyridine solution was removed by filtration and the glass beads rapidly washed with 50 volumes of 2 ~ NaCl solution at p~l 10. The glass beads were then washed with 50 volumes of distilled water.
Sulfation of the material could be readily confirmed by a modi~ication of the colorimetric method described by Smith et al ~P.X. Smith, A. X. Mallia and G. T. Hermanson, Anal.
3s Biochem. lO9, 466 - 473, 1980).

, :, ~ ,. ' ,;
. .

The ability of the sulfated polyhyroxysilica gel to adsorb coagulation factors IX a~d X was tested. To 10 ml aliquots of a solution of redissolved PTC powder (0.6% w/v) in 0~05 M sodium citrate pH 6.5 containiny 0.1 M NaCl was added 2 5 grams of either the sulfated or the non-sulfated polyhydroxy silica gel. The suspension was mixed on a tube rocker for 15 minutes after which the suspension was centrifuged at low speed to pellet the gel. The presence of F. IS and F~ X activities was assayed in the respective o supernatants as well as in ~he starting PTC solution and are listed in Table 1.

Table 1 lS Factor X and IX Activities in PTC Solution Adsorbed with Sulfated and Non-sulfated Polyhydroxysilica F. IX ~/ml F. X U/ml Starting PTC solution 2.6 13.4 20 Supernatant of adsorption with non-sulfated poly~
hydroxy silica 5.1 13.4 Supernatant of adsorption with sulfated polyhydroxy silica 1.3 5.9 As is evident from the data in the Table, the supernatant of the adsorption with the sulfated gel contains considerably less of both F. IX and F. X activity than the staxting PTC solution. Some activation of F. IX is apparent after contact with the non-sulfated material.
Nevertheless, the data confirm that the sulfated poly-hydroxysilica gel possesses the ability data confirm that .the sulfated polyhydroxysilica gel possesses the ability to .35 adsorb coagulation factors in apparent contrast to the non-sulfated form.

: :

B. ~se of Sulfa~ed Trisacryl in preparation of partially purified coagulation factors~

EXAMPLE 1.
5 Use of Sulfated Trisacryl for the Preparation of a Partially Purified Factor IX from PTC
by Gradient Elution (3001 - 34) PTC powder, a dried, vitamin K dependent coagulation factor-containing material which is an in~rmediate s~ep in the commercial process for ~he production of Factor IX
- concentrate, was redissolved at 1~ w/v in 0.05 M sodium citrate containing 0.1 M NaCl, pH 6.5. After dissolution ; of the powder, the solution was adsorbed with powdered, fumed silica (Aerosil 380, a product of DeGussa) at a 1 w/v ratio for 45 minutes at 42 C. The suspension was centrifuged and the insoluble pellet of Aerosil and adsorbed materials was discarded. The supernatant was filtered and cooled to 10 C. The chi].led solution was 20 applied to a column (17 cm x 14 cm) containing sulfated Trisacryl and equilibrated in 0.05 M sodium citrate containing 0.1 M NaCl at pH 6.5. Column flow rates were approximately 10 L/hr. Material not binding to the column and eluting as a breakthrough peak was discarded. The 2S applied sample was followed by a volume of equilibration buffer sufficient to reduce the protein in the eluate ~o an A280 ~0.1. A 40 liter gradient of NaCl from 0.1 M to 0.6 M
was then applied to the column and 1 liter fractions collected and analyzed for their protein content and

3~ immunologically detectable amounts of coagulation Factors IX and X as well as Protein C.

The elution profile of the column is shown in ~i~ure 1.
Fractions containing immunologically identifiable amounts 3s of Factors IX, X and Protein C are indicated in the Figure.
Protein C and Factor X were poorly resolved and eluted ~ Tî~e~or~

~ . ., .

early in the gradient at a sodium chloride concentration of approximately 0.15 - 0.3 M. Factor IX was observ~d to elute later in the gradient at approximately 0.4 M NaCl although there was some overlap with F. X.

The Factor IX-containing regions of the elution of similar column runs were pooled and dialyzed vs. 0.015 M sodium citrate containing 0.12 M NaCl. After concentration and sterile filtration, a partially purified F. IX preparation was obtained for animal studies with the following characteristics:

Factor IX activity 30.2 units/ml Protein concentration 4.41 mg/ml 15 F. IX spec. activity 6.85 units/mg NAPTT 1:10 163 sec 1:100 312 sec EXAMPLE 2.
Rapid Chromatography of DEAE Eluate on Sulfated Trisacryl with Quantitation of Factors II, VII, IX and X

A concentrate containing vitamin K-dependent clotting factors was obtained by contacting Effluent I plasma with 2s DE~E Sephadex and eluting the adsorbed gel as follows, After removal of the spent Effluent I, the DEAE gel was sequentially washed with ~1) 0.2 M sodium bicarbonate, ~2) 0.3 M sodium bicarbonate, and (3) 0.2 M sodium chloride containing 0.01 M sodium citrate, pH 6.5, The Factor IX
30 and other vitamin K-dependent fa~tors were then eluted from the DEAE gel with a buffex containing O.055 ~ NaCl and 0.01 M sodium citrate, p~l 6.5. The eluate was diafiltered against six volume exchanges of 0,05 M sodium citrate containing 0.1 M NaCl ! pH 6.5. 73 L of eluate were 3s ob ained and aliquots were frozen for ~ubsequent chromato-graphic studies. The content of clotting factors in the ~ T~A~th)~rl~

-~ CL-114 ,, :
~ .

., .

~ 3 g dialyzed eluate expressed as total units is given in Table .

Table 2 sContent of Vitamin K-dependent Clotting Factors in the Dialy~ed Eluate from DEAE Sephadex~
Contact of Eluate I

Units/ml To~al Units 10 Factor II (prothrombin) 12.0 876,000 Factor VII 2.5 182,130 Factor IX - 6.6 481,800 Factor X 12.8 919,800 Protein C positive for antigen lS Protein S- positive for antigen For the purpose of determining maximum flow characteristics of the sulfated trisacryl gel, a column of the following dimensions was constructed: height ~ 5 cml `, 20 diameter = 25 cm. B.7 liters of th DEAE elute described above was treated with Aerosil 380 at 0.25% w/v at 5 C for 60 minutes. The insoluble Aerosil was removed by filtra-tion and the clariied eluate applied to the sulfated trisacryl column at 24 liters/hour. ~Flow rates of up to 2S 60 liters per hour were obtained with this column configuration without visible deformation of gel or marked loss of column performance~. The elution profile of the column is shown in Figure 2. After application of the clarified DEAE elute, the column was washed with 0.1 M
NaCl, 0.05 M sodium citrate, pH 6~5. Separate step elutions were then carried out at 0.275 M NaCl and 0.55 M
NaCl respectively for differential elution of clotting factors. Both of the latter elution steps included 0.05 M
sodium citrate, pH 6.5. Table 3 describes the presence of 3s various clotting factors in each purification step.

7~r~1en~o~r 1~

.
.

~ 3~3 Table 3 Quantitation of Clotting Factors in Purification Scheme Outlined in Example 2 ~ Total Uni~s Presence of Antigen Step F IX F X F. II P.C.
DEAE eluate 57420 145,290 Post Aerosil DEAE
eluate 57936 119,950 ~ +
Unbound column peak 5076 43,990 + +
0.275 M NaCl peak25376 ~0,512 - +
0.55 M NaCl peak 14076 1,173 lS While the separations obtained in this example are not optimum, this chromatography is illustrative of the fact that resolution of vitamin X-dependent factors i5 possible at very high flow rates. Of particular note is the obser-vation that Factor II (prothrombin) elutes in the unbound eluate and is not detectable in later elution steps. This was confirmed in elute and is not detectable in later elution steps. This was confirmed in similar chromato-graphic runs using specific F. II clotting assays. Protein C antigen is observed to co-chromatograph with F. X~ The recovery of F. X and F~ IX activities in the various elution steps is in good agreement with the total amounts of respective activities applied to the column.

EXAMPLE 3.
Stepwise Elution of Clotting Factors from Sulfated Trisacryl with Emphasis on the Removal of F. X from the F. IX

To a column of sulfated trisacryl with the dimensions described in Example 1 was applied 5 liters of a solution of DEAE eluate prepared as described in Example 2.

: CL-114 :' g3 N~ Aerosil adsorption of this solution was carried out.
Rather the sodium chl~ride concentration of the applied DEAE eluate was adjusted to 0.3 M pxior to application.
The column was also pre-equilibrated in 0.3 M NaCl, 0.05 M
5 sodium citrate, pH 6.5. After application of the sample, the column was washed with equi~ibration buffer to reduce the A280 of the column eluate to ~0.2. The Factor IX was then eluted in a single step with a buffer containing 0.55 M NaCl. The recoveries of Factors IX and X are given in 10 Table 4.

Table 4 Recoveries of Factors IX and X in the Sulfated Trisacryl Chromatography of Example 3 F. IX F. X
Step A280 units/ml total u. units/ml ~ ~ u.
DE~E eluate 5.41 4.9 24,580 14.5 72,500 (S liters)*
20 Unbound peak 1.22 0.59 10,500 4.1 72,980 (17.8 L) 0.55 M NaCl - 0.60 1.75 15,120 undete~ble eluted p~ak (8.64 L) This example demonstrates that it is possible to effec-tively remove F. X from the final F. IX preparation as eluted from the sulfated trisacryl. Since Factor X is, in our experience, the most troublesome and most likely contaminant ~rom among the other vitamin K-dependent 30 factors, it follows that the levels of other clotting factors such as F. II are also likely to be very low în the F. IX peak described here. Indeed, in other ~imilar chromatographies, prothrombin was fuund to be undetectable in F. IX peaks.

, . . . ~ } -

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A sulfated, synthetic, noncarbohydrate affinity chromatography gel matrix adsorbing agent comprising a sulfatable polymer, said sulfatable polymer selected from the group consisting of a synthetic organic affinity chromatography gel matrix prepared by polymerization of at least one poly-merizable organic monomer, and polymeric silica particles, said sulfatable polymer having been treated by a process consisting essentially of treat-ing with a sulfating agent so as to provide a multi-plicity of sulfate groups bound directly to the polymer backbone.

2. An affinity chromatography gel matrix adsorbing agent according to claim 1, wherein said sulfatable polymer is said synthetic organic affinity chromatography gel matrix prepared by polymerization of at least one polymerizable organic monomer and is selected from the group consisting of sulfated poly-hydroxylated acrylates and methacrylates and sulfated polyvinylbenzene.

3. An affinity chromatography gel matrix adsorbing agent according to claim 2, wherein said sulfated polyhydroxylated acrylate is obtained by copolymerization of the monomer, N-acryloyl-2-amino-2-hydroxymethyl-1,3-propane diol.

4. In a method of isolating and purifying at least one of the group of blood coagulation factors and proteins selected from the group consisting of factors IX, X, II, VII, VIII, protein C, protein S, prothrombin and tissue plasminogen activator, from a source of said blood coagulation factors and proteins selected from blood, blood plasma, plasma concentrates and tissue culture fluids, obtained by means of bio-technology techniques, by the steps of (a) contacting the blood, blood plasma, plasma concentrates and tissue culture fluids with at least one affinity chromatography adsorbing agent, (b) separating the non-adsorbed solution from the non-adsorbing agent, and (c) eluting the adsorbed blood coagulation factors and proteins from the adsorbing agent using an eluant effective to elute the desired factor(s) and protein(s), the improvement comprising using as the adsorbing agent a sulfated, synthetic, non-carbohydrate affinity chromaography gel matrix adsorbing agent com-prising a sulfatable polymer, said sulfatable polymer selected from the group consisting of (i) a synthetic organic affinity chromatography gel matrix prepared by polymerization of at least one polymerizable organic monomer, and (ii) polymeric silica particles, said sulfatable polymer having been treated by a pro-cess consisting essentially of treating with a sul-fating agent so as to provide a multiplicity of sul-fate groups bound directly to the polymer backbone.

5. A method according to claim 4, wherein said sulfatable polymer is said synthetic organic affinity chromatography gel matrix prepared by polymerization or at least one polymerizable organic monomer and is selected from the group consisting of sulfated poly-hydroxylated acrylates and methacrylates and sulfated polyvinylbenzene.

6. A method according to claim 5, wherein the polymer backbone of said sulfated polyhydroxylated acrylate is obtained by copolymerization of the monomer, N-acryloyl-2-amino-2-hydroxymethyl-1,3-propane diol.

7. A method according to claim 4, including the step of treatment to render said blood coagulation factors and proteins non-infective by infectious viruses, including hepatitis virus, bacteria and retroviruses, including the AIDS-causing agent(s).